MUTATIONS REVEAL G PROTEIN ACTIVATION MECHANISM

异源三聚体G蛋白将细胞表面受体的信号传递给 细胞内效应器。最近解决的阿尔法的晶体结构 GDP和GTP结合形式的转导蛋白亚单位(Galphat)使 Galphat是研究G蛋白信号转导机制的理想模型。 这个项目的目标是确定受体的结构基础。 G蛋白的激活，通过扰乱GDP的释放通过位点- 定向诱变，同时筛选重组Galphat 作为研究终止机制的工具的分子 G蛋白的作用。 为了研究视网膜视紫红质受体对G蛋白的激活作用， 将在受体偶联和GDP结合区引入突变 基于这种蛋白质的高分辨率结构。加尔法特 加速GDP释放的突变将结构性地激活 蛋白质和模拟受体催化激活。对这些问题的分析 突变将确定负责耦合的结构元素 G蛋白的受体。 这些释放GDP的突变也将被用来探索 管理Galphat活动的终止。因为稳态GTP 这种突变蛋白的水解率不受国内生产总值的限制。 释放后，其GTPase活性可用于检测GTPase的激活 蛋白质(GAP)在没有激活视紫红质的情况下。美国政府的角色 Galphat效应器、cGMP磷酸二酯酶(PDE)和其他潜在的缺口 将会被调查。

Heterotrimeric G proteins relay signals from cell-surface receptors to intracellular effectors. Recently solved crystal structures of the alpha subunit of transducin (Galphat) in both GDP- and GTP-bound forms make Galphat an ideal model for studying the mechanism of G protein signaling. The goals of this project are to define the structural basis of receptor activation of G protein, by perturbing release of GDP through site- directed mutagenesis, and at the same time to select recombinant Galphat molecules to serve as tools for studying the mechanism for terminating the action of G proteins. To study G protein activation by the receptor, retinal rhodopsin, mutations will be introduced in receptor coupling and GDP binding regions of Galphat, based on high resolution structures of this protein. Galphat mutations that accelerate GDP release will constitutively activate the protein and mimic receptor catalyzed activation. Analysis of these mutations will identify structural elements responsible for coupling receptors to G proteins. These GDP-releasing mutations will also be used to probe mechanisms that regulate termination of the activity of Galphat. Because steady-state GTP hydrolysis by such mutant proteins is not limited by the rate of GDP release, their GTPase activities can be used to detect GTPase Activating Proteins (GAPs) in the absence of activated rhodopsin. The role of the Galphat effector, cGMP phosphodiesterase (PDE), and other potential GAPs will be investigated.